The goal of the proposed research is to understand the multifaceted mechanisms that control the activity of two skeletal muscle sarcoplasmic reticulum Ca2+ release channels, the ryanodine receptors RyR1 and RyR3. The RyR ion channels are composed of four RyR 560 kDa peptide subunits and various associated proteins with a total molecular mass of >2,500 kDa. RyR1 and RyR3 conduct monovalent and divalent cations, and numerous endogenous effectors ranging from divalent cations (Ca2+ and Mg2+) to small molecules (e.g. ATP) and proteins (e.g. FK506 binding protein, triadin, calsequestrin, calmodulin,) regulate RyR function and muscle function. The RyRs are also targets for redox active molecules such as glutathione, oxygen tension and NO. The principal hypothesis to be tested in the proposed research is that there are different regulatory domains that confer ion conductance and signaling functions to the RyRs. The specific aims are to : (1) Characterize the regulation of the skeletal muscle RyRs by redox active molecules and identify RyR regulatory redox-sensitive cysteines by mass spectrometric analysis and mutagenesis. (2) Identify regions in the RyRs that transduce the functional effects of calmodulin, and characterize genetically modified mice that have mutations in the calmodulin-binding domain of the RyRs. (3) Test the role of the RyR associated proteins triadin, junction and calsequestrin by gene silencing. (4) Test the hypothesis that negatively charged amino acid residues support the high rates of ion flux by the RyRs. The functional properties of normal and mutant RyRs will be determined in intact cells, isolated membranes and purified channels in contractility, Ca2+ imaging, SR vesicle-Ca2+ flux, [3H]ryanodine binding and single channel measurements. The outlined studies will help to define the regulatory mechanisms of Ca2+ release from sarcoplasmic reticulum and provide an understanding of how these processes are altered during impaired muscle function and in RyR-associated central core disease.